1. Field of Invention
The present invention relates to a one-step immunoassay method and immunoassay kit for measuring percentage of hemoglobin A1c in a sample.
2. Related Art
Hemoglobin A1c (HbA1c) is a glycated hemoglobin formed by binding between the amino group of the N-terminal valine of the .beta.-chain of hemoglobin and glucose aldehyde group resulting first in a Schiff's base and further a stable ketoamine by Amadori Rearrangement. Since percentage of HbA1c reflects mean blood sugar content prior to 1 to 2 months, it is well accepted for blood sugar level management.
So far, an instrument performing measurement by high performance liquid chromatography (HPLC) using ion exchange chromatography, namely a glycohemoglobin analyzer, has been primarily used for the measurement of HbA1c % in blood. Although this method gives excellent producibility, it has also been pointed out that several disadvantages exist, including requirement of a specialized HPLC analyzer system, limitations of processing capability, and inaccurate results with other forms of hemoglobin. In addition, it requires highly technical maintenance.
In recent years, HbA1c measurement techniques other than the HPLC method have been developed to solve the above mentioned problems, and such reagents have been marketed. Immunoassay in particular has been applied for the measurement of HbA1c % due to its high specificity.
Sandwich assay, for example, using two antibodies, has been considered for use as an immunoassay for HbA1c. On the other hand, adsorption-immunoassay, wherein hemoglobins including HbA1c in a sample are physically adsorbed to a solid phase and the adsorbed HbA1c is detected with an antibody having high specificity for HbA1c, is well known. In this method hemoglobins are directly adsorbed to a solid phase, and adsorbed HbA1c is considered to be in a proportion equal to the proportion in hemoglobins when excess hemoglobins are applied to the limited surface of the solid phase. Since HbA1c is expressed in the clinical field as a percentage (%) relative to the total amount of hemoglobins, the percentage of HbA1c in the blood can be determined without measuring the absolute amount of HbA1c nor of total hemoglobins, when the percentage of HbA1c in the adsorbed hemoglobins on a solid phase is in a proportion equal to that in blood. In this method it is required for hemoglobins to be adequately adsorbed on the solid phase. Sequential steps are, therefore, carried out in this method. Namely, hemoglobins are first adsorbed on the surface of a solid phase such as latex particles or polystryrene beads, the resulted solid phase is then washed or separated to remove unadsorbed materials as necessary, and the HbA1c adsorbed on the solid phase is specifically detected by an anti-HbA1c antibody that specifically binds HbA1c. Enzyme immunoassay (EIA) reagents using this method are also commercially available.
In this two-step assay using a commercially available EIA kit, since a washing procedure is required between the first adsorption step and the second immunoreaction step, the procedure is more complex and requires longer processing times as compared with a simultaneous or one step immunoassay. In addition, there is also a possibility of the hemoglobin adsorbed onto the solid phase being released during the washing steps. Moreover, in these types of kits, since the glycated N-terminal residue of the hemoglobin .beta.-chain is used for the reaction with specific antibody to HbA1c, there are many situations which require strong denaturing conditions to expose the glycated N-terminal residues during or prior to adsorption on the solid phase. However, since there is a high possibility that the enzyme antibody conjugate may be inactivated under these conditions, a simultaneous adsorption-immune reaction step may not be applied due to possible inactivation of enzyme and/or antibody of enzyme labeled anti-HbA1c antibody. Due to the strong denaturing conditions used in such commercially available kits, a denatured blood sample must be used immediately after its preparation, because variation in the measured values occurs due to the formation of precipitates and so forth, thus preventing the obtaining of correct values. Consequently, this method is not considered to be suitable for processing large numbers of samples.
On the other hand, immunoassay has also been applied to latex agglutination instead of EIA. A hemolyzed specimen is added to an unsensitized latex suspension to adsorb the hemoglobins on the surface of latex, then the mouse monoclonal antibody specific to HbA1c is added followed by the addition of anti-mouse IgG polyclonal antibody for the resulting agglutination wherein the degree of agglutination reflects HbA1c (%) in a sample. Although this method does not require a washing step, since a large amount of latex is required to adsorb hemoglobins, there is a possibility of the occurrence of non-specific agglutination of latex resulting in the appearance of abnormally high values or the risk of difficulty in detecting increased agglutination caused by antibody-antigen reaction of HbA1c.
Diabetes and other adult related diseases are steadily increasing with increase in the elderly population. Accompanying this trend, since diagnostic parameters such as HbA1c % which is widely recognized as an indicator of blood sugar status is the object of diagnosis when examinations are required to be performed on elderly persons, it is necessary to improve specimen processing capacity. There is therefore a tremendous need for the development of diagnostic reagents that enable processing to be performed easily, accurately and in large volume. The currently available HPLC method has a low specimen processing capacity and specialized instrumentation requirement with complexity in maintenance. On the other hand, the currently available EIA and latex agglutination methods require complex procedures and give inaccurate results, for example, because of the several reasons mentioned above.
As the result of conducting studies on developing a method enabling measurement of HbA1c % in a sample easily and accurately on the basis of immunoassay with high specificity, the inventors of the present invention constructed a method that allows measurement of HbA1c % in a sample using a one-step procedure in which adsorption of hemoglobins on the solid phase and reacting of antigen HbA1c and anti-HbA1c antibody are performed simultaneously, thereby leading to completion of the present invention.
Namely, the present invention discloses a method for measuring HbA1c % comprising the steps of simultaneously bringing a sample containing HbA1c in contact with a solid phase and an anti-hemoglobin A1c antibody resulting in the formation of adsorbed HbA1c bound to an antibody, separating the bound anti-hemoglobin A1c antibody to the solid phase from unbound materials, and detecting the amount of bound anti-hemoglobin A1c antibody or the unbound anti-hemoglobin A1c antibody.
In addition, the present invention discloses a one-step immunoassay kit for measuring hemoglobin A1c % in a sample comprising a solid phase and an anti-hemoglobin A1c antibody.